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First published online 7 December 2004
doi: 10.1242/jcs.01562

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Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3

¹ Department of Biochemistry, McMaster University, Hamilton, Ontario, L8S 4LB, Canada
² Henderson Research Centre, Hamilton, Ontario, L8V 1C3, Canada
³ Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, L8S 4LB, Canada
⁴ Department of Medicine, McMaster University, Hamilton, Ontario, L8S 4LB, Canada

View larger version (14K): [in a new window]   Fig. 1. Pretreatment with low concentrations of valproate increases resistance to ER stress-induced cell death. (A) HepG2 cells were cultured in the presence of various concentrations of valproate as indicated. Lactate dehydrogenase (LDH) activity was determined in media after 48 hours. The plot represents LDH released after exposure to valproate as a percentage of the total LDH activity recovered from control cells lysed with 0.5% Triton X-100. Data are presented as the mean±s.d. of three separate experiments. (B) LDH release assay measuring cell death in HepG2 cells pretreated with 0 mM () or 0.5 mM () valproate for 18 hours and then exposed for 6 hours to the calcium ionophore, A23187 (0-10 µM), tunicamycin (0-40 µg/ml) or H2O2 (0-0.5%) as indicated. Results are expressed as the percentage of the total LDH present in untreated cells and depicted as mean±s.d. from three separate experiments. *P<0.01 when compared to levels in the corresponding controls. (C) Determination of caspase 3 activation in HepG2 cells pretreated for 18 hours in the absence or presence of 0.5 mM valproate and then exposed to 0 or 20 mg/ml tunicamycin for 6 hours as indicated. Hatched bars represent cells that were treated with the caspase 3 inhibitor, Z-VAD, in addition to tunicamycin. Caspase 3 activity was determined in the cell lysates and normalized to total protein concentrations. Data are presented as the mean±s.d. of three separate experiments. *P<0.01 when compared to the activity in the corresponding control.

View larger version (58K): [in a new window]   Fig. 2. Valproate blocks ER stress-induced activation of SREBP and accumulation of free cholesterol in HepG2 cells. (A) Filipin staining of HepG2 cells pre-cultured for 2 hours in the presence or absence of 0.5 mM valproate and then exposed to 5 µM A23187, 2 µg/ml tunicamycin or 5 mM glucosamine as indicated. As a control, HepG2 cells were exposed to the HMG CoA reductase inhibitor, mevastatin (50 µM) and 5 mM glucosamine as described above. After 24 hours, cells were washed, fixed in paraformaldehyde and stained with filipin. Intracellular filipin-cholesterol complexes were visualized by fluorescence microscopy and images were captured with a digital camera. Representative images are shown. (B) Median fluorescence of filipin-stained cells treated as described in A. *P<0.01 when compared with the fluorescence in the corresponding control. **P<0.05 compared to the relative fluorescence under the same conditions without valproate. (C) Immunoblot analysis of mature SREBP-2 protein levels in HepG2 cells pretreated with 0.5 mM valproate for 2 hours and then exposed to 0, 1 or 5 mM glucosamine for 24 hours as indicated. Total protein lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes and immunostained with antibodies against SREBP-2.

View larger version (30K): [in a new window]   Fig. 3. Valproate induces the expression of ER-resident chaperones. (A) Relative levels of GRP78/BiP and GADD153/CHOP mRNA normalized to GAPDH mRNA as determined by northern blot analysis. HepG2 cells were cultured for the indicated times in the presence of 0.5 mM valproate. Total RNA was resolved by agarose gel electrophoresis, transferred to nylon membranes and subjected to blot hybridization with 32P-radiolabeled cDNA probes encoding human GRP78/BiP and GADD153/CHOP. Relative mRNA levels were quantified using a Typhoon 9410 phosphoimaging system and normalized to GAPDH (loading control) RNA levels. (B) Immunoblot analysis of protein chaperone levels in HepG2 cells cultured for the indicated time in the presence of 0.5 mM valproate. Total protein lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes and immunostained with antibodies against GRP78/BiP, HSP47, calreticulin, PDI or HSP70 as indicated. As a loading control, an identical blot was immunostained with an antibody against ß-actin.

View larger version (66K): [in a new window]   Fig. 4. Valproate does not induce an ER stress response in HepG2 cells. (A) Protein synthesis rates measured by the incorporation of [35S]methionine/cysteine into proteins during a 15-minute pulse of labeling of HepG2 cells that were pre-exposed to 10 µM A23187 or 0.5 mM valproate for the indicated times. Total protein extracts were resolved by SDS-PAGE and visualized by Coomassie Blue staining (top panel) or autoradiography (middle panel). Total 35S incorporation in each of the tested conditions was determined by scintillation counting and plotted as a percentage of the control count (bottom panel). (B) Immunoblot analysis of protein extracts treated as described in A. Total protein lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were immunostained with antibodies against total eIF2 or the ser-52 phosphorylated form of eIF2. (C) Immunoblot analysis of HepG2 cells cultured for the indicated times in the presence of 0.5 mM valproate or in the presence of 10 µg/ml tunicamycin or 10 µM A23187 for 8 hours. Total protein lysates were resolved and transferred to nitrocellulose membranes. Membranes were immunostained with antibodies against total GADD153/CHOP, ATF4 or ß-actin (loading control). (D) Analysis of ERdj4 mRNA levels in HepG2 cells cultured for the indicated times in presence of 0.5 mM valproate or for 8 hours with 10 µg/ml tunicamycin. Total RNA was resolved by agarose gel electrophoresis, transferred to nylon membranes and subjected to blot hybridization with 32P-radiolabeled cDNA probes encoding ERdj4 and GAPDH (loading control).

View larger version (44K): [in a new window]   Fig. 5. Valproate pretreatment does not abrogate an ER stress response. (A) Northern blot analysis of HepG2 cells pretreated for 0, 24 or 48 hours with 0.5 mM valproate and then challenged with 0, 2 or 10 µg/ml tunicamycin (left panel) or 0, 2, 10 µM A23187 (right panel) for 4 hours as indicated. Total RNA was resolved by agarose gel electrophoresis, transferred to nylon membranes and hybridized to radiolabeled cDNA probes encoding human GRP78/BiP or GADD153/CHOP. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe. (B) Immunoblot analysis of HepG2 cells treated with valproate, tunicamycin and A23187 as described above. Total protein lysates were resolved and transferred to nitrocellulose membranes. Membranes were immunostained with antibodies against GADD153/CHOP, the ser9-phosphorylated form of eIF2 (eIF2-P) or total eIF2 as indicated.

View larger version (24K): [in a new window]   Fig. 6. Effects of ER stress-inducing agents and valproate on GSK3/ß activity. (A) GSK3 and -ß activity immunoprecipitated from cells exposed to 10 µg/ml tunicamycin or 10 µM A23187 for 2 hours. Quantitative values are expressed as a percentage of GSK3 or -ß activity in untreated cells. Data are expressed as the mean±s.d. (n=3). (B) Recombinant GSK3 and -ß activity in the presence of 0-5 mM valproate or 0-50 mM lithium. Quantitative values are expressed as a percentage of recombinant GSK3 or -ß activity in the absence of inhibitor. Data are expressed as the mean±s.d. (n=3). *P<0.05 relative to activity in the corresponding control. (C) GSK and -ß activity immunoprecipitated from HepG2 cells. Valproate (0.5 mM) was added in vivo to the cells 2 hours prior to harvesting and immunoprecipitation or to the immunopurified GSK3 during the kinase assay as indicated. Quantitative values are expressed as a percentage of GSK3 or ß activity in untreated cells. Data are expressed as the mean±s.d. (n=3). *P<0.05 relative to levels in the corresponding control.

View larger version (38K): [in a new window]   Fig. 7. Agents that inhibit GSK3 activity also block ER stress-induced lipid accumulation and apoptosis. (A) Determination of caspase 3 activity in HepG2 cells pretreated for 1 hour in the presence or absence of 20 mM LiCl and then exposed to 0 or 20 µg/ml tunicamycin for 6 hours as indicated. Caspase 3 activity was determined in cell lysates and normalized to total protein concentrations. Data are presented as the mean±s.d. of three independent experiments. *P<0.01 when compared to the corresponding control. (B) Filipin staining of HepG2 cells pretreated for 1 hour in the presence of 0.5 mM valproate, 20 mM LiCl or 20 µM GSK inhibitor II (GSKin) and then challenged with 0 or 5 mM glucosamine as indicated. After 24 hours, cells were washed, fixed in paraformaldehyde and stained with filipin. Intracellular filipin-cholesterol complexes were visualized by fluorescence microscopy. (C) Median fluorescence of filipin-stained cells treated as described in B. *P<0.01 when compared to the corresponding control. **P<0.01 compared to relative fluorescence in the absence of GSK3 inhibition.

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